Sphingomonas Strains Producing Greatly Increased Yield of PHB-Deficient Sphingan (Diutan)

ABSTRACT

PHB-deficient  Sphingomonas  strains having improved sphingan yield are provided. Certain of the  Sphingomonas  strains are diutan-producing strains that exhibit a dramatic improvement in productivity and yield due to a combination of certain genetic modifications that affect PHB and sphingan synthesis. Moreover, the sphingans produced from such strains have superior characteristics including improved filterability, clarity, and improved rheology-modifying characteristics. The sphingans provided are, thus, highly desirable in a variety of commercial and industrial uses, including personal care items, cement applications, and oilfield applications.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of U.S. application Ser.No. 13/733,933, which is a divisional application of U.S. applicationSer. No. 12/533,649 filed Jul. 31, 2009, which are hereby incorporatedby reference in their entireties.

SEQUENCE LISTING

The sequence listing in the file named “68492o705000.txt” having a sizeof 179,295 bytes that was created Jul. 31, 2009 is hereby incorporatedby reference in its entirety.

BACKGROUND

1. Field of the Art

This application generally relates to the construction of PHB-deficientSphingomonas strains that produce high yields of diutan with improvedfilterability. In another aspect, this application relates to diutanproduced from PHB-deficient Sphingomonas strains that produce highyields of diutan with improved filterability.

2. Description of Related Art

A number of bacteria of the genus Sphingomonas produce polysaccharidescalled sphingans that have related structures with a generally conservedtetrasaccharide backbone structure and different side chains (ref. no.1, 6, 7, 8, 10). The sphingans gellan, welan, rhamsan and diutan areproduced commercially for use in food, oilfield or personal careapplications. The value of sphingan polysaccharides lies in theirabilities to modify the rheology of aqueous solutions, i.e., to thickenliquids, suspend solids, stabilize emulsions, or form gels and films.

Sphingans are structurally related to one another, but are notidentical. Common members of the genus Sphingomonas and the sphingansthey produce include Sphingomonas elodea ATCC 31461, which producesgellan (S-60); Sphingomonas sp. ATCC 31555, which produces welan(S-130); Sphingomonas sp. ATCC 31961, which produces rhamsan (S-194);Sphingomonas sp. ATCC 53159, which produces diutan (S-657); Sphingomonassp. ATCC 31554, which produces an as yet unnamed polysaccharide (S-88);Sphingomonas sp. ATCC 31853, which produces an as yet unnamedpolysaccharide (S-198); Sphingomonas sp. ATCC 21423, which produces anas yet unnamed polysaccharide (S-7); Sphingomonas sp. ATCC 53272, whichproduces an as yet unnamed polysaccharide (NW-11); Sphingomonas sp.FERM-BP2015 (previously Alcaligenes latus B-16), which produces alcalan(Biopolymer B-16) and the like. A description of the Sphingomonads andthe polysaccharides they produce can be found, for example, in U.S. Pat.Nos. 4,377,636; 4,326,053; 4,326,052 and 4,385,123 (for ATCC 31461 andits S-60 polysaccharide); in U.S. Pat. No. 4,342,866 (for ATCC 31555 andS-130); in U.S. Pat. No. 4,401,760 (for ATCC 31961 and S-194); in U.S.Pat. No. 5,175,278 (for ATCC 53159 and S-657); in U.S. Pat. Nos.4,331,440 and 4,535,153 (for ATCC 31554 and S-88); in U.S. Pat. No.4,529,797 (for ATCC 31853 and S-198); in U.S. Pat. No. 3,960,832 (forATCC 21423 and S-7); in U.S. Pat. No. 4,874,044 (for ATCC 53272 andNW-11); in U.S. Pat. No. 5,175,279 (for FERM BP-2015 and B-16), each ofwhich is incorporated by reference herein in its entirety to the extentthat they are not inconsistent with the disclosure herein.

One particular sphingan, diutan (also known as heteropolysaccharideS-657), is prepared by fermentation of strain Sphingomonas sp. ATCC53159 (ref. no. 17). Diutan exhibits unique rheological properties inaqueous solutions including high thermal stability, superior suspensionproperties, and the ability to generate high viscosity at lowconcentrations. The diutan polysaccharide imparts significantpseudoplasticity to polar solvents such as water, such that diutan canact as a rheological modifier that is capable of particle suspension,friction reduction, emulsion and foam stabilization, filter cakedeposition and filtration control. Consequently, diutan has foundindustrial utility as a rheological modifier in a variety of contexts,including cementitious systems as disclosed in U.S. Pat. No. 6,110,271,which is incorporated herein by reference in its entirety to the extentthat it is not inconsistent with the disclosure herein.

Diutan consists of a repeat unit with a backbone comprised of[→4)-α-L-rhamnose-(1→3)-β-D-glucose-(1→4)-β-D-glucuronicacid-(1→4)-β-D-glucose-(1→] and a two-sugar L-rhamnose side-chainattached to the (1→4) linked glucose residues (ref. no. 2, 7). TwoO-acetyl groups are attached per repeat unit to the 2′ and 6′ positionsof the (1→3) linked glucose (ref. no. 4).

Progress has been made in elucidating the genetics and biochemistryunderlying biosynthesis of diutan and other sphingans. Genes forbiosynthesis of sphingans S-88, S-7, and gellan have been identified(ref. no. 5, 12, 13, 15). Genes for several glycosyl transferases of thebackbone structure have been analyzed biochemically (ref. no. 11, 14),as have genes gelC and gelE, potentially involved in chain lengthdetermination (ref. no. 9). Several of the genes for synthesis of sugarnucleotide precursors have also been elucidated (ref. no. 12). Thegenetics and biochemistry of polymerization, secretion and control ofpolysaccharide molecular length are less defined.

A cluster of genes involved in biosynthesis of diutan has beenidentified that includes genes for glycosyl transferases, genes encodingenzymes for synthesis of a precursor molecule dTDP rhamnose, and genesfor secretion of the polysaccharide (ref. no. 3). Plasmids, e.g., pS8and pX6, containing some of the genes in the aforementioned cluster,were shown to increase the yield of diutan by about 10%, and one plasmidin particular (pS8) was found to significantly improve the rheologicalproperties of diutan from the wild-type strain (ref. no. 18).

Growth conditions typically used for producing diutan and othersphingans also promote production of the internal storage polymerpolyhydroxybutyrate (“PHB”), which is generally regarded as anundesirable side-product and is difficult to remove during sphinganpreparation. The PHB can form small insoluble particles that interferewith clarity and filterability, limiting the usefulness of thesphingans. For example, the turbidity imparted by PHB particles canlimit applicability for household and personal care products in whichappearance is critical for consumer acceptance. Moreover, certainoilfield uses require filterability; however, the PHB particles can plugsmall pores in oil field rock formations, preventing the flow of thesphingan solution and/or the return flow of the crude oil after treatingthe well. Finally, as PHB synthesis and sphingan synthesis compete forthe available carbon source, PHB synthesis can have some adverse effecton sphingan yield.

Accordingly, attempts have been made to eliminate PHB production insphingan-producing strains. Ref. no. 26 describes a strain ofSphingomonas elodea (a gellan-producing species) that was isolatedfollowing chemical mutagenesis. This strain, called LPG-2, has decreasedPHB production, but produces gellan of inconsistent quality and yield.

A more targeted approach to eliminating PHB production was undertaken bydeletion of a gene required for PHB synthesis, the phaC gene (ref. no.20). Precise deletion of phaC from a diutan producing strain (ATCC53159) reproducibly resulted in poor growth and severely reduced diutanproductivity (strains NPD3 and NPD6). These strains exhibit increasedcarbohydrate hydrolysis and accumulation of organic acids, suggesting acritical role for phaC in maintaining normal cellular metabolism.Derivatives with less impaired diutan productivity were subsequentlyisolated. Two independent derivatives, PDD3 and PDD6, haveuncharacterized spontaneous mutation(s) and remain PHB-deficient (ATCCdeposit nos. PTA-4865 and PTA-4866, respectively). Though recovery of upto 90% of total diutan yield has been reported (ref. no. 20), this yieldwas only obtained following a greatly increased culture growth time andhas not been consistently reproducible. Under standard growthconditions, diutan productivity and yield by these strains is onlyapproximately half of wild-type levels.

SUMMARY

In view of the foregoing, there is a need to overcome the low sphinganproductivity that is characteristic of PHB-deficient strains. Thepresent disclosure addresses this need in the art by providing agenetically modified strain of Sphingomonas which not only lacks PHBproduction but also provides surprisingly high diutan productivity.Unexpectedly, the plasmids pS8 and pX6—which give only modestimprovement in diutan productivity in PHB-producing strain—are now shownto greatly improve diutan productivity in a PHB-deficient strain. Thegreat improvement in diutan productivity was particularly surprisingbecause the plasmids contain genes involved in diutan biosynthesis andare not known to contain any genes that would offset the metabolicdeficiency of a PHB-deficient strain. Certain embodiments of thesegenetically modified strains, described infra, fully overcome the pooryield and low productivity of PHB-deficient strains, whilesimultaneously attaining the desired filterability and clarity ofPHB-deficient sphingans.

Certain embodiments encompass a mutant strain of the genus Sphingomonashaving a genetic modification that reduces, or, preferably,substantially or entirely eliminates the production of PHB. In exemplaryembodiments, the genetic modification inactivates the phaA gene, phaBgene, phaC gene, or any combination thereof. In another exemplaryembodiment, the genetic modification to impair PHB synthesis is obtainedby screening or selection for a PHB-deficient organism. The geneticmodification that impairs PHB synthesis can reduce or completelyeliminate PHB production, and can optionally be conditional, such asconditional induction, suppression, overexpression, knock-out, etc. of agene involved in PHB synthesis, a gene that suppresses PHB synthesis, orany combination thereof. Optionally, a mutant strain of the genusSphingomonas having a genetic modification that reduces, or, preferably,substantially or entirely eliminates the production of PHB also includesat least one additional genetic modification that suppresses the poorgrowth and/or poor diutan productivity of such strains. In an exemplaryembodiment, the additional genetic modification can include at least oneof the suppressor mutations contained in strains PDD3, PDD6, or both, ora variant of such suppressor mutation(s).

Certain embodiments encompass a method of increasing sphingan productionin a host organism, such as an organism of the genus Sphingomonas.Exemplary methods of increasing sphingan production include increasingthe expression in the host organism of at least one gene involved insphingan synthesis. Such genes can be involved in sphingan synthesis,secretion, polymerization, synthesis of precursors, control ofpolysaccharide molecular length, etc. For example, additional copies ofat least one gene involved in sphingan production can be introduced onan extrachromosomal element (such as a plasmid) or can be integratedinto the host genome, or both. Such genes can be derived from the hoststrain or can be homologs derived from another species or strain.Homologs can include functional, structural, or sequence homologs of agene involved in sphingan production or of a gene having an enzymaticactivity the same as or similar to a gene involved in sphingansynthesis. In exemplary embodiments, the genes can be obtained byscreening or selection for a Sphingomonas strain having increasedsphingan production. Exemplary methods of increasing sphingan productionalso include introduction of genes involved in sphingan productionhaving modified (non-native) sequences, such as modified promoter orenhancer elements, expression-optimized sequences, etc. Additionally,the native chromosomal copy of at least one gene involved in sphingansynthesis can optionally be deleted, or be replaced by any of theforegoing.

In certain embodiments, an extrachromosomal or integrated sequenceelement containing at least one gene, such as all of the genes that arecontained in the insert in plasmid pS8 and/or pX6, or homolog(s)thereof, can be introduced into a Sphingomonas strain. For example, theat least one gene can include dpsS, dpsG, dpsR, dpsQ, dpsI, dpsK, dpsL,dpsJ, dpsF, dpsD, dpsC, dpsE, dpsM, dpsN, atrD, atrB, dpsB, rmlA, rmlC,rmlB, rmlD, orf7, orf6, orf5, or any combination thereof. In certainexemplary embodiments, the gene(s) include at least one gene encoding asphingan biosynthetic enzyme, such as a dpsG polymerase. In anotherexemplary embodiment, such genes encoding a sphingan biosynthetic enzymecan include a dpsG polymerase and a glucose-1-phosphatethymidylyltransferase gene; a dTDP-6-deoxy-D-glucose-3-5-epimerase gene;a dTDP-D-glucose-4,6-dehydratase gene; and adTDP-6-deoxy-L-mannose-dehydrogenase gene. In another exemplaryembodiment, such genes encoding a sphingan biosynthetic enzyme caninclude a dpsG polymerase and a rhamnosyl transferase IV gene; abeta-1,4-glucuronosyl transferase II gene; a glucosyl isoprenylphosphatetransferase I gene; and a glucosyl transferase III gene. In anotherexemplary embodiment, such a gene encoding a sphingan biosyntheticenzyme can include a dpsG polymerase and one or more of thepolysaccharide export genes dpsD, dpsC, and dpsE. In another exemplaryembodiment, such a gene encoding a sphingan biosynthetic enzyme caninclude a rhamnosyl transferase IV gene; a beta-1,4-glucuronosyltransferase II gene; a glucosyl isoprenylphosphate transferase I gene;glucosyl transferase III gene; a glucose-1-phosphatethymidylyltransferase gene; a dTDP-6-deoxy-D-glucose-3-5-epimerase gene;a dTDP-D-glucose-4,6-dehydratase gene; and adTDP-6-deoxy-L-mannose-dehydrogenase gene. In another exemplaryembodiment, such a sphingan biosynthetic enzyme can be selected from thegroup consisting of a gene encoding a polymerase; lyase; rhamnosyltransferase IV; beta-1,4-glucuronosyl transferase II; glucosyltransferase III; polysaccharide export protein; secretion protein;glucosyl-isoprenylphosphate transferase I; glucose-1-phosphatethymidylyltransferase; dTDP-6-deoxy-D-glucose-3-5-epimerase;dTDP-D-glucose-4,6-dehydratase; dTDP-6-deoxy-L-mannose-dehydrogenase,and any combination thereof. In certain embodiments, any combination ofthe foregoing genes or homologs thereof can be introduced into aSphingomonas strain. In one exemplary embodiment, the Sphingomonasstrain is a diutan-producing strain, such as ATCC 53159, or aPHB-deficient derivative thereof, such as a phaC deletion strain, suchas NPD3, NPD6, PDD3, or PDD6. In another exemplary embodiment, theSphingomonas strain is derived from Sphingomonas elodea ATCC 31461,Sphingomonas sp. ATCC 31555, Sphingomonas sp. ATCC 31961, Sphingomonassp. ATCC 53159, Sphingomonas sp. ATCC 31554, Sphingomonas sp. ATCC31853, Sphingomonas sp. ATCC 21423, Sphingomonas sp. ATCC 53272,Sphingomonas sp. FERM-BP2015, or a PHB-deficient derivative, such as aphaC deletion strain of any of the foregoing, or a phaC deletion strainbearing further mutation(s) that improve growth or sphinganproductivity. In an exemplary embodiment, the phaC deletion strain isderived from a gellan-producing strain, such as LPG-2 (ref. no. 26),NPG-1, NPG-2, NPG-3, PDG-1, PDG-3 (ref. no. 20) or a derivative thereof.

In exemplary embodiments, a gene involved in sphingan synthesis can bederived from a homolog of a gene contained in plasmids pS8 or pX6. Sucha homolog can be a Sphingomonas homolog, i.e., derived from an organismof the genus Sphingomonas. Exemplary organisms from which Sphingomonashomologs can be derived include Sphingomonas elodea ATCC 31461,Sphingomonas sp. ATCC 31555, Sphingomonas sp. ATCC 31961, Sphingomonassp. ATCC 53159, Sphingomonas sp. ATCC 31554, Sphingomonas sp. ATCC31853, Sphingomonas sp. ATCC 21423, Sphingomonas sp. ATCC 53272,Sphingomonas sp. FERM-BP2015, or any combination thereof. In anotherexemplary embodiment, a gene involved in sphingan synthesis can encode apolypeptide having at least about 70% sequence identity, such as about75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%,about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, orabout 100% sequence identity, to a polypeptide sequence of SEQ ID NO: 3,5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41,43, 45, 47, 49, 51, or 53. In another exemplary embodiment, a geneinvolved in sphingan synthesis can be encoded by a polynucleotide havingat least about 60% sequence identity, such as about 65%, about 70%,about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%,or about 100% sequence identity, to a polynucleotide sequence of SEQ IDNO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36,38, 40, 42, 44, 46, 48, 50, or 52.

Certain embodiments of the present compositions include a diutan,particularly a PHB-deficient diutan, exhibiting an improvement (relativeto diutan produced from a wild-type strain) in a number of differentviscosity measurements. Among these are: i) an intrinsic viscosity ofgreater than about 150, preferably higher than about 155, morepreferably higher than about 160 dL/g; ii) a sea water 3 rpm viscositygreater than about 35, such as greater than about 37, such as greaterthan about 40, such as greater than about 42, such as greater than about45, such as greater than about 47, such as greater than about 50 dialreading; iii) a sea water 0.3 rpm viscosity greater than about 35,000,such as greater than about 39,000, such as greater than about 40,000,such as greater than about 42,000, such as greater than about 45,000,such as greater than about 48,000, such as greater than about 50,000,such as greater than about 54,000 centipoise (cP); and a PEG low shearrate viscosity greater than about 3500, such as greater than about 3700,such as greater than about 3900, such as greater than about 4000, suchas greater than about 4200, such as greater than about 4500, such asgreater than about 4700, such as greater than about 5000, such asgreater than about 5200, such as greater than about 5500, such asgreater than about 5700, such as greater than about 6000 cP.

Certain embodiments of the present strains include a mutant strain ofthe genus Sphingomonas that is able to produce PHB-deficient diutan at arate of at least about 0.10 g/L/hr, such as at least about 0.11 g/L/hr,such as at least about 0.12 g/L/hr, such as at least about 0.13 g/L/hr,such as at least about 0.14 g/L/hr, such as at least about 0.15 g/L/hr,such as at least about 0.2 g/L/hr, and/or a yield of PHB-deficientdiutan of at least about 12 g/L, such as at least about 15 g/L, such asat least about 16 g/L, such as at least about 17 g/L, such as at leastabout 18 g/L, such as at least about 19 g/L, such as at least about 20g/L, such as at least about 21 g/L. For example, certain embodiments caninclude a mutant strain of the genus Sphingomonas able to producePHB-deficient diutan at a rate of between about 0.15 g/L/hr and about0.60 g/L/hr, such as between about 0.16 g/L/hr and about 0.5 g/L/hr,such as between about 0.17 g/L/hr and about 0.4 g/L/hr, such as betweenabout 0.18 g/L/hr and about 0.35 g/L/hr, such as between about 0.19g/L/hr and about 0.3 g/L/hr, such as between about 0.2 g/L/hr and 0.25g/L/hr, such as between about 0.21 g/L/hr and about 0.22 g/L/hr.Additionally, certain embodiments can include a mutant strain of thegenus Sphingomonas able to produce a yield of PHB-deficient diutanbetween about 12 g/L and about 30 g/L, such as between about 13 g/L andabout 25 g/L, such as between about 14 g/L and about 22 g/L, such asbetween about 19 g/L and about 21 g/L.

Certain embodiments of the present strains include a mutant strain ofthe genus Sphingomonas containing a genetic modification thatsubstantially or entirely eliminates the production of PHB and a geneticmodification that results in increased production of a sphingan, whereinthe mutant strain of the genus Sphingomonas increases the rate ofproduction or yield of PHB-deficient diutan by at least about 50%, suchas by at least about 60%, such as by at least about 70%, such as by atleast about 80%, such as by at least about 90%, such as by at leastabout 100%, such as by at least about 110%, such as by at least about120%, such as by at least about 120%, such as by at least about 130%,such as by at least about 140% relative to a congenic strain containingthe genetic modification that substantially or entirely eliminates theproduction of PHB and lacking the genetic modification that increasesthe production of a sphingan. For example, the increase in the rate ofproduction or yield of PHB-deficient diutan can be between about 50% andabout 200%, such as between about 60% and about 190%, such as betweenabout 70% and about 180%, such as between about 80% and about 170%, suchas between about 90% and about 160%, such as between about 100% andabout 150%, such as between about 110% and about 140%, such as betweenabout 120% and about 130%.

In certain embodiments, one or more copies of specific DNA sequences areintroduced within certain Sphingomonas strains to provide increasedbiosynthetic production of high viscosity diutan polysaccharide that isessentially free of PHB. The engineered bacteria containing such genesfor increased production produce significantly greater amounts ofPHB-deficient diutan polysaccharide compared to non-engineered bacteriaand create diutan with the aforementioned resultant high viscosityproperties.

The DNA can be delivered into bacteria of the genus Sphingomonas inmultiple copies (via plasmid, other known manner) or increasedexpression of the genes via a suitable method, e.g., coupling to astronger promoter. After insertion of the DNA into the target bacteria,the production of diutan can be determined by fermenting the engineeredbacteria and comparing the yield in terms of amount produced and qualityproduced. Increased production and viscosity can both be determined bycomparison with other diutan-producing strains.

Sphingomonas strains, such as the genetically modified strains describedherein, can be used to produce sphingans, such as diutan, byfermentation. Generally, a suitable medium for fermentation is anaqueous medium which contains a source of carbon (for example,carbohydrates including glucose, lactose, sucrose, maltose ormaltodextrins), a nitrogen source (for example, inorganic ammonium,inorganic nitrate, urea, organic amino acids or proteinaceous materials,such as hydrolyzed yeast, soy flour or casein, distiller's solubles orcorn steep liquor), and inorganic salts. A wide variety of fermentationmedia will support the production of diutan according to the presentinvention. One of ordinary skill in the art can readily determine anappropriate media formulation.

Carbohydrates can be included in the fermentation broth in varyingamounts—usually between about 1 and 10% by weight (preferably 2-8%) ofthe fermentation medium. The carbohydrates can be added prior tofermentation or, alternatively, during fermentation. The amount ofnitrogen can, for example, range from about 0.01% to about 0.4% byweight of the aqueous medium. A single carbon source or nitrogen sourcecan be used, as well as mixtures of these sources. Among the inorganicsalts which are useful in fermenting Sphingomonas bacteria are saltswhich contain sodium, potassium, ammonium, nitrate, calcium, phosphate,sulfate, chloride, carbonate and similar ions. Trace metals, such asmagnesium, manganese, cobalt, iron, zinc, copper, molybdenum, iodide andborate, can also be advantageously included in the broth.

In certain embodiments of the present method, Sphingomonas strainsundergo fermentation. Fermentation can be carried out, for example, attemperatures between about 25 degrees C. and 40 degrees C., preferablybetween about 27 degrees C. and 35 degrees C. An inoculum can beprepared by standard methods of volume scale-up, including shake flaskcultures and small-scale submerged stirred fermentation. The medium forpreparing an inoculum can be the same as the production medium or can beany one of several standard media well-known in the art, such as Luriabroth or YM medium. More than one seed stage can be used to obtain thedesired volume for inoculation. Typical inoculation volumes range fromabout 0.5% to about 10% of the total final fermentation volume.

Certain embodiments of the present methods include agitation of thefermentation medium. In some embodiments, an agitator is containedwithin a fermentation vessel, whereby the contents of the agitationvessel are mixed. The vessel also can have automatic pH and foamingcontrols. The production medium can be added to the vessel andsterilized in place, e.g., by heating. Alternatively, the media can besterilized separately before addition. A previously grown seed culturecan be added to the cooled medium (typically at the preferredfermentation temperature of about 27 degrees to about 35 degrees C.),and the stirred culture can be fermented for about 48 to about 110hours, producing a high viscosity broth. The sphingan, such as diutan,can be recovered from the broth by, for example, a standard method ofprecipitation with an alcohol, generally isopropanol.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows greatly improved diutan productivity of PHB-deficientstrains bearing plasmids pX6 and pS8 relative to PHB-deficient strainswithout the plasmids.

FIG. 2 shows greatly improved diutan yield from PHB-deficient strainsbearing plasmids pX6 and pS8 relative to PHB-deficient strains withoutthe plasmids.

FIG. 3A illustrates poor filterability of a PHB-containing diutanpreparation (0.04% S657/pS8 diutan in seawater). FIG. 3B illustratespoor filterability of an independent PHB-containing diutan preparation(0.04% S657/pS8 diutan in seawater).

FIG. 4A illustrates improved filterability of a PHB-deficient diutanpreparation (0.04% PDD3/pS8 diutan in seawater). FIG. 4B illustratesimproved filterability of a PHB-deficient diutan preparation (0.04%PDD3/pS8 diutan in seawater). FIG. 4C illustrates improved filterabilityof a PHB-deficient diutan preparation (0.04% PDD3/pS8 diutan inseawater).

FIG. 5 presents a map showing the inserts contained in plasmids pS8 andpX6.

FIG. 6 shows the insert sequence contained in plasmid pS8 (SEQ ID NO:1).

FIG. 7 shows the insert sequence contained in plasmid pX6 (SEQ ID NO:54).

DETAILED DESCRIPTION

Two PHB-deficient bacterial strains derived from Sphingomonas sp. ATCC53159 (S657) were previously developed and designated PDD3 and PDD6 (seeref. no. 20). These strains exhibit approximately half of the diutanproductivity of the wild-type strain (S657). The plasmid pS8 containsseveral genes involved in diutan biosynthesis in a multicopy plasmid andhas been used to enhance diutan productivity and rheology (ref. no. 18).See also refs. no. 21-23 which describe the use of plasmid mediated geneamplification to increase polysaccharide yield (DNA segments and methodsfor increasing polysaccharide production).

As is shown in greater detail below, applicants have now shown thatintroduction of the plasmids pX6 and pS8—which contain multiple genesinvolved in diutan biosynthesis, but are not known to contain any genesthat would offset the metabolic deficiency of a PHB-deficientstrain—into PHB-deficient mutants PDD3 and PDD6 results in an unexpectedsignificantly improved productivity (g/L/hr) and dry weight yield (g/L)of the PDD strains (70% to >100% increase) relative to the PHB-deficientstrains without the introduced plasmids. The PHB-deficient strainsproduced fewer cells and no PHB, thus, more of their dry weight yield isdiutan polysaccharide. Due to their increased productivity, thesestrains can be used for more economical production of PHB-deficientdiutan than strains lacking these genetic modifications. Moreover, aclarified diutan produced from such strains exhibits improvedfilterability and clarity due to the absence of PHB particles relativeto PHB-containing diutan. Such PHB-deficient diutan can be particularlydesirable in a variety of applications, including household and personalcare products, cementitious systems, for enhanced oil recovery,fracturing, well bore clean-up and other ‘pay zone’ applications, or anyother application involving particle suspension, friction reduction,emulsion and foam stabilization, filter cake deposition and filtrationcontrol, or modification of the rheology of aqueous solutions (such asto thicken liquids, suspend solids, stabilize emulsions, or form gelsand films, etc.). Additionally, upon acid hydrolysis, the PHB-deficientdiutan leaves little to no residue as compared to PHB-containing diutan.The low acid hydrolysis residue renders the PHB-deficient diutanparticularly suitable in oil field applications, such as fracturing, inwhich a viscosifying fluid is degraded after fracturing the formation,so the return flow of oil is maximized. Unlike the PHB-containingdiutan, which contains PHB particles that would plug the pores in therock formation, PHB-deficient diutan would not plug the pores in theformation, leading to improved oil yield.

In one exemplary embodiment of the present strains, a plasmid containingthe relevant DNA sequence is inserted into a recipient Sphingomonasbacterium and replicates in the recipient cell, typically giving one orseveral (at least two and usually 4-10) copies of the DNA segment thatresult in increased production of high viscosity diutan polysacchariderelative to a strain lacking the DNA sequence. Alternatively or inaddition to insertion of a plasmid-borne DNA sequence, DNA sequencesthat integrate into the bacterial chromosome can also be used. The useof conjugation or mobilization to transfer DNA into recipient bacteriais generally effective. Electroporation or chemical transformation ofcompetent cells with purified DNA can also be used. Other vectors orbacteriophages can be used to transfer DNA into the host cell.Maintaining the DNA segments on plasmids (or other well known deliveryvectors) in the recipient diutan-producing Sphingomonas is notnecessary. It is routine to introduce additional copies of a DNA segmentinto the bacterial chromosome so that the segments are replicated eachgeneration by the same mechanism that replicates the bacterial DNA.Alternative to or in conjunction with methods that increase the copynumber of a DNA sequence, increased gene expression can be achieved byusing stronger promoter elements.

The following terms shall be used throughout the specification inconnection with the present invention and have the meaning indicated:

The term “Sphingomonas” is used throughout the specification to refer tostrains of gram-negative bacteria from the genus Sphingomonas.

The term “inserted” is used throughout the specification to describe theprocess and outcome of transferring DNA into a Sphingomonas strain. Suchisolated DNA can be introduced first into, as one non-limitingpossibility, a desired plasmid (such as pLAFR3), by well-knowntechniques in the art, and then transferred, for example, by conjugationor mobilization into a recipient Sphingomonas bacterium.

The term “gene amplification” is used to refer to either increasedcopies of genes, for example, by cloning the target genes on a multicopyplasmid (such as from 4 to 10 copies) or by insertion of multiple copies(such as from 4 to 10) of the genes into the bacterial genome, oralternatively, increased expression of genes by modification of promoterelements to increase gene expression. Both of these methods and otherscan result in increased amounts of the encoded proteins.

The term “biosynthesis” is used throughout the specification to describethe biological production or synthesis of a sphingan by Sphingomonasbacteria.

Cloning of DNA in the present invention relies on general techniques andmethods which have become standard in the art. It is noted that anynumber of methods can be used to clone DNA segments according to thepresent invention, and the present invention is not limited, forexample, to the use of plasmid cloning vectors. For example, DNAfragments can be cloned by insertion into a bacteriophage vector. Incertain embodiments of the present methods, cloned DNA sequences areintroduced to a Sphingomonas strain via a plasmid or other deliveryvector.

The term “ectopic promoter” is used to refer to a non-native promoter,i.e., a promoter with some sequence difference(s) relative to the nativepromoter. Such a promoter can be, for example, a strong promoter whichdrives a measurably increased level of transcription relative to thenative promoter. An ectopic promoter can also be a regulated promoter,whereby gene expression is increased or decreased in response to somefactor, such as a small molecule, temperature, presence of a geneproduct, etc. Suitable promoters for a particular use are well known inthe art.

The term “genetic modification” is used throughout the specification torefer to a genetic change. Generally, a genetically modified organism,such as a Sphingomonas strain, is described with reference to a “parent”strain which does not contain the genetic modification. Exemplarygenetic modifications include those that increase, decrease, or abolishthe expression of a gene. Such changes include modification ofchromosomal and extrachromosomal genetic material. Exemplary geneticmodifications include introduction of a plasmid, deletion orsubstitution of a chromosomal sequence. For example, a chromosomal genecan be inactivated by a targeted deletion of part or all of the codingsequence and/or regulatory element (e.g., as described in ref. no. 20),or genetic screen, optionally including mutagenesis (e.g., as describedin ref. no. 26). Chromosomal genetic modification can also involve atargeted replacement, e.g., to replace a native gene promoter with aninducible promoter, regulated promoter, strong promoter, etc.Chromosomal gene modification can also involve gene amplification, i.e.,introduction of at least one additional copy of at least one gene.Extrachromosomal genetic material can be introduced, for example, on aplasmid, which can be single-copy, multi-copy, or high-copy, as is wellknown in the art. Genetic modification can be coupled to a selectablemarker, such as an antibiotic resistance gene, which helps ensure thatthe genetic modification is retained.

The term “essentially free of PHB” is used throughout the specificationto refer to a composition, such as a sphingan (e.g., diutan), having agreatly reduced PHB content when compared to a similar compositionprepared from a wild-type or PHB-containing strain. Great reduction canbe at least a 90% reduction, 95% reduction, 99% reduction, 99.5%reduction, etc. in PHB content (where PHB content is expressed as afraction of the dry weight of the sphingan composition). Suitable assaysfor measuring PHB content include the 15% HCl solubility and residuetest, HPLC, gas chromatography, and gas chromatography coupled to massspectrometry (GC-MS). In certain embodiments of the presentcompositions, a clarified (e.g., cellulase clarified) diutan preparationthat is essentially free of PHB can yield less than approximately 1%,such as less than approximately 0.5%, such as less than approximately0.1%, residue in a 15% HCl solubility and residue test.

The term “PHB-deficient diutan” is used throughout the specification torefer to a diutan produced from a PHB-deficient strain, such as strainbearing a genetic modification inactivates the phaA gene, phaB gene,phaC gene, or any combination thereof.

The term “phaC gene” is used throughout the specification to refer to aphaC gene of a Sphingomonas strain. Examples of phaC gene sequences areprovided in (ref. no. 20); however, other phaC gene orthologs are alsoencompassed except where the context indicates otherwise.

When an amount, concentration, or other value or parameter is given as alist of upper preferable values and lower preferable values, this is tobe understood as specifically disclosing all ranges formed from any pairof an upper preferred value and a lower preferred value, regardless ofwhether ranges are separately disclosed.

The term “a” or “an” as used herein means “one” or “one or more”.

The term “about” or “approximately” as used herein means within anacceptable error range for the particular value as determined by one ofordinary skill in the art, which will depend in part on how the value ismeasured or determined, i.e., the limitations of the measurement system.For example, “about” can mean within 1 or more than 1 standarddeviations, per practice in the art. Where particular values aredescribed in the application and claims, unless otherwise stated, theterm “about” means within an acceptable error range for the particularvalue.

Unless otherwise indicated, all numbers expressing quantities ofingredients, properties such as molecular weight, reaction conditions,and so forth used in the specification and claims are to be understoodas being modified in all instances by the term “about” Accordingly,unless indicated to the contrary, the numerical parameters set forth inthe following specification and attached claims are approximations thatmay vary depending upon the desired properties sought to be obtained bythe present invention. At the very least, and not as an attempt to limitthe application of the doctrine of equivalents to the scope of theclaims, each numerical parameter should at least be construed in lightof the number of reported significant digits and by applying ordinaryrounding techniques.

Except wherein indicated otherwise, all measurements and protocols areconducted at standard temperature and pressure, i.e., approximately 20°C. and approximately 1 atmosphere. Except where indicated otherwise,“sea water 3 rpm viscosity,” “sea water 0.3 rpm viscosity,” and “lowshear rate viscosity in the presence of polyethylene glycol” aremeasured as described in Example 2 (paragraphs [0065]-[0066]), below.

The invention will now be described in more detail with respect to thefollowing, specific, non-limiting examples.

EXAMPLES Example 1 Production of Diutan

This example described an increased yield of PHB-deficient diutanproduced from several genetically modified Sphingomonas strains.

Methods

The plasmids pS8 and pX6 were transferred into PHB-deficientSphingomonas strains PDD3 and PDD6 by triparental conjugal mating asdescribed previously (ref. no. 3) and which is well known in the art.Strains PDD3, PDD6, 5657, and S657/pS8 are as described previously (ref.nos. 17, 18, and 20). Strains PDD3/pS8, PDD6/pS8, PDD3/pX6, PDD6/pX6,PDD3, PDD6, S657, and S657/pS8 were grown in 15 L volumes in 20 LApplikon fermentors with agitation and aeration. For the plasmidcontaining strains, the antibiotic tetracycline at 5 mg/L was addedthroughout the fermentation to ensure retention of the plasmid. KOH wasadded as needed to control pH. Two seed stages were used with 1% to 6%inoculum transfers. The fermentation media contained corn syrup ascarbohydrate source, an assimilable nitrogen source, and salts.

At the end of the fermentation, each broth was treated by introductionof glucoamylase enzyme to hydrolyze any remaining oligosaccharides fromthe corn syrup. The viscosities of the fermentation broths were measuredvia a Brookfield® viscometer run at 60 rpm with a spindle #4. The diutangums produced were then precipitated from an aliquot of broth with twovolumes of isopropyl alcohol. The diutan fibers were collected on afilter and dried. For some strains, multiple replicates were prepared,and the results presented below are the average values across thesereplicates.

Results

The presence of a plasmid containing genes involved in diutan synthesis(pX6 or pS8, see FIG. 5) greatly improved the diutan production byPHB-deficient strains compared to the parent PHB-deficient strains PDD3and PDD6. Diutan productivity of PHB-deficient strains was greatlyimproved by between 70% and 142% (FIG. 1 and Table 1B) relative to theparental strains. This increased productivity was much greater than forthe wild-type (S657) strain with the introduced pS8 plasmid, whichdemonstrated increased productivity by only 33%. Three of the fourPHB-deficient, plasmid-containing strains had higher productivity thanthe wild-type (S657) strain, and strain PDD3/pX6 had productivityessentially equal to S657/pS8. Diutan yield of PHB-deficient strainsbearing these plasmids was also greatly improved by between 53% and 90%(FIG. 2 and Table 1B) relative to the parental strains. This increasewas much greater than for the wild-type (S657) strain with theintroduced pS8 plasmid, which only increased diutan yield by 18%.

Consistent with these results, the PHB-deficient strains also exhibitedincreases in final broth viscosity due to introduction of the plasmids,indicating greater diutan content. The PHB-deficient, plasmid-containingstrains also had lower cell density (measured by OD₆₀₀) than thewild-type strain with or without plasmid pS8 (Table 1B), indicating thatthe unclarified products from these strains are expected to contain ahigher proportion of diutan (due to the presence of fewer bacterialcells). Due to the higher purity of the diutan produced fromPHB-deficient, plasmid containing strains (both due to lower cellcontent and absence of PHB), the extent of productivity and yieldimprovement in these strains compared to wild-type strains is likely tobe even greater than these measurements indicate.

TABLE 1A Final culture conditions. Final Cell Final Broth ReplicatesDensity Viscosity Strain PHB (n) (OD₆₀₀) (cP) S657 (wild-type) + 2 8.223000 S657/pS8 + 2 5.84 3775 PDD3 − 1 3.80 3000 PDD3/pX6 − 1 4.14 3650PDD3/pS8 − 3 3.50 4158 PDD6 − 1 4.15 2500 PDD6/pX6 − 2 3.73 3125PDD6/pS8 − 3 3.74 3783

TABLE 1B Diutan productivity and yield. Percent Percent Percent ChangePercent Change Change over Change over Over PHB- Over PHB- Produc- S657defi- S657 defi- tivity (wild- cient Yield* (wild- cient Strain PHB(g/L/hr) type) parent (g/L type) parent S657 + 0.153 17.5 (wild-type)S657/pS8 + 0.203 +33% 20.7 +18% PDD3 − 0.082 −46% 11.4 −35% PDD3/pX6 −0.197 +140% 20.9 +83% PDD3/pS8 − 0.177 +116% 21.7 +90% PDD6 − 0.067 −56% 9.4 −46% PDD6/pX6 − 0.114  +70% 14.4 +53% PDD6/pS8 − 0.162 +142% 17.3+84% *Total dry weight of unclarified precipitate (dry weight yield).

Example 2 Diutan Analysis

The diutan samples produced in the method of Example 1 were analyzed foruses as oilfield additives for oil recovery and for uses requiring goodsuspension and stabilization (such as for cement additives for waterretention and quick set-up).

Methods

The oilfield industry relies on a “sea water viscosity” (SWV) test as anindicator of acceptable performance for rheology modifiers in oilrecovery. This test indicates whether a rheology modifier cansufficiently increase viscosity in briny conditions of sea water, suchas those encountered in seabed oil recovery. Typically, a sea waterviscosity test employs synthetic seawater produced by mixing 419.53grams of sea salt (ASTM D-1141-52) per 9800 grams of deionized water.For a seawater viscosity test, a rheology modifier is dispersed insynthetic seawater by vigorous mixing (e.g., 35 minutes at approximately11,500 rpm in a Fann Multimixer (Model 9B5, part number N5020)). Thesample is cooled to approximately 25° C. before the viscosity ismeasured. For a 3-rpm viscosity test, the sample is placed on the Fannsample platform (Fann model 35 A; Torsion spring MOC 34/35 F0.2b; BobB1; Rotor R1) and the speed is adjusted to 3 rpm by turning the motor tolow speed and setting the gearshift in the middle position. The readingis then allowed to stabilize, and the shear stress value is read fromthe dial and recorded as the SWV 3 rpm dial reading (DR). For the0.3-rpm reading, a Brookfield viscometer is used (Brookfield LV DV-II orDV-III viscometer, with LV-2C spindle) to measure the viscosity. Thespeed of the spindle is set to 0.3 rpm, and the spindle is allowed torotate at least 6 minutes before the viscosity is recorded as theSWV-0.3 rpm reading and expressed in centipoises (cP).

The LSRV test (a low shear rate viscosity using polyethylene glycol asdispersant as described below) is a general test for viscosity at a lowshear rate. Typically, the higher the viscosity the better a sample isat stabilization and suspension. For example, in a cementitiousapplication, a higher viscosity in the LSRV test indicates that a diutanshould help suspend particulates in the cement more effectively, givinga more homogeneous cement/concrete, thus, providing better strength anddurability. The LSRV test measures the viscosity of a 0.25% solution ofbiogum in Synthetic Tap Water (STW). STW is prepared by adding 10.0grams NaCl and 1.47 grams CaCl₂.2H₂O to 10 liters of deionized water.For the viscosity measurement, 0.75 grams of biogum is added to 4.5grams Polyethylene Glycol 200 (CAS 25322-68-3) in a 400-mL beaker andthoroughly dispersed. Then, 299 grams of STW are added to the beaker andmixed for approximately 4 hours using a low-pitched, propeller-stylestirrer at 800±20 rpm. After the 4-hr mixing time, the beaker is placedin a 25° C. water bath and allowed to sit undisturbed for approximately30 minutes. The viscosity is then measured using a Brookfield LVviscometer equipped with a 2.5+ torque spring (or equivalent instrument,such as Model DVE 2.5+) at 3 rpm using the LV 1 spindle after allowingthe spindle to rotate for 3 minutes and expressed in centipoises (cP).

Results

The diutan samples produced in Example 1 above were analyzed todetermine suitability for use in cement and oilfield applications (Table2). Utility for stabilization and suspension, such as for cementadditives for water retention and quick set-up, was evaluated by lowshear rate viscosity (LSRV) testing. Suitability for oil recovery wasevaluated using sea water viscosity (SWV) tests at 0.3 rpm and 3 rpm asan indicator of the effectiveness of a gum to increase viscosity inbrines.

In the LSRV test, diutan produced from PHB-deficient strain PDD3containing either plasmid performed better than or about equal to thewild-type strain bearing pS8, with greater improvement observed forPDD3/pX6 than for PDD3/pS8 (Table 2). In the SWV test at 0.3 rpm, diutanproduced from plasmid-containing PHB-deficient strains derived from PDD3performed better than wild-type strains bearing pS8. In the SWV test at3 rpm, either PHB-deficient strain bearing pS8 performed essentiallyequally to the wild-type strains bearing pS8. Together, these resultsindicate that a PDD3/pS8 diutan is particularly suitable for oilfieldapplications and cement applications.

TABLE 2 Rheology of unclarified PHB-deficient diutan. SWV SWV DiutanLSRV 0.3 rpm 3 rpm Produc- (centi- (centi- (dial Strain PHB tivity npoise) n poise) n reading) S657 + ++ 1 5110 2 37,400 2 40.0 S657/pS8 ++++ 1 6610 2 48,600 2 56.5 PDD3 − + 1 3160 — nt. — nt. PDD3/pX6 − +++ 16910 1 54,400 1 45.5 PDD3/pS8 − +++ 2 6198 3 51,867 2 57.0 PDD6 − + 13020 — nt. — nt. PDD6/pX6 − +++ 2 5188 2 41,600 1 43.0 PDD6/pS8 − +++ −nt. 1 38,400 1 57.0 nt.: Not tested.

Example 3 Low Acid Residue of PHB-Deficient Diutan Methods

The indicated strains were grown in 1000 gallon fermentors and inmultiple Applikon® fermentors to prepare larger samples for testing andanalysis. After the fermentations had finaled, the broths were eitherleft untreated or enzyme clarified using one of two methods.

The first method, clarification with a cellulase, CELLUCLAST™(“Clarified”) was as follows: First, the broth temperature was adjustedto 50° C. Next, the pH was adjusted to between 5.0 and 5.4. CELLUCLAST™enzyme (1 g/L) was then added, and the broth was incubated for twohours. Stock solutions of EDTA and Lysozyme in distilled water were thensequentially added to the broth to a final concentration of 0.25 g/LEDTA and 0.05 g/L Lysozyme, and the broth incubated for one hour. The pHwas then adjusted to 8.0 to 8.5. Protex 6 L protease was then added tothe broth at a final concentration of 0.5 g/L and the broth wasincubated for two hours. Finally, the diutan gum was precipitated byaddition of three volumes of isopropyl alcohol, dried, and milled.

The second enzyme clarification (“Treated”) was similar to the firstmethod, except the initial pH adjustment and the addition of CELLUCLAST™enzyme were omitted.

Dried diutan samples were analyzed using the 15% HCl Solubility andResidue Test, as follows: 1.6 grams of a sample is rehydrated in 253 mlSynthetic Tap Water (typically 1 hr mixing at 1000 rpm). The mixingspeed was then decreased to 500 RPM, and 147 mL of concentrated HCl(37%) is added to the rehydrated sample and mixed for 10 minutes. Thesample container was then sealed and incubated at 150 degrees F. fortwenty-four hours. The sample was again mixed, then a 100 gram aliquotwas removed. The aliquot was quantitatively transferred to a Gelmanfilter apparatus containing a 0.5 micron filter. The filter was dried,cooled, and weighed prior to filtration and again after filtration. Theweight of residue was reported as a percentage of the dry weight ofpolymer in the 100 gram aliquot (dry weight is determined by drying asample of the same starting material).

Results

The acid residue test measures the amount of insoluble material thatremains in a sample after acid hydrolysis. Low acid residue is preferredfor certain uses, for example, an oilfield use in which the diutan isremoved by acid hydrolysis and any insoluble residue has the potentialto clog pores in the formation. This residue test also provides anindirect indication of the amount of PHB in a diutan preparation becausethe acid residue of a wild-type diutan is predominantly PHB. For aPHB-deficient diutan, the acid residue indicates an upper bound for thePHB content.

Results of the acid residue test are provided in Table 3, with residueindicated as a percentage of the starting sample material. Unlike thePHB-containing strains, which contained between 1.8 wt % and 6.8% wt %acid residue, the clarified PHB-deficient strain produced only 0.05 wt %acid residue. These results confirmed that the PHB-deficient strainproduced diutan that would not damage an oilfield formation and,moreover, that the PHB-deficient diutan contains less than 0.05% PHB byweight.

TABLE 3 Low acid residue of clarified PHB-deficient diutan. Strain PHBWeight Percentage Residue S657/pS8 + 1.80% (Treated) S657 + 6.78%(untreated) S657/pS8 + 2.98% (untreated) PDD3/pS8 − 0.05% (Clarified)

Example 4 Confirmation that PHB is Absent from PHB-Deficient DiutanMethods

The analytical method measured the PHB content of diutan preparationsand can also be used to measure the PHB content of otherpolysaccharides. In this method, the diutan is digested with an aqueoushypochlorite solution leaving the PHB intact; the PHB polymer is thenhydrolyzed, then esterified to the propyl ester; and finally, theresulting ester is measured by gas chromatography with flame ionizationdetection. The instrument used was the Hewlett Packard Model 6890 GasChromatograph System equipped with a HP model 7673 auto injector, flameionization detector, and Hewlett Packard HP 5MS column (30 m×250 μm×0.25μm nominal id).

The detailed protocol is as follows. Approximately 35-40 mg of eachdiutan sample was weighed into a glass centrifuge tube, in duplicate andthe weight recorded to the nearest 0.1 mg. Approximately 5 mL ofapproximately 5% sodium hypochlorite (JT Baker Cat #4616 or equivalent)was then added to each tube and the tubes vortexed. Samples were thenincubated at approximately 37° C. for 12-18 hours, resulting inhypochlorite digestion. Tubes were then centrifuged at approximately8000 rpm for approximately 40 minutes, and the hypochlorite supernatantswere removed with a disposable pipette and discarded. Samples were thenwashed twice by addition of 5 mL deionized water with centrifugation andsupernatant removal as in the previous step. Samples were thenevaporated to dryness under reduced pressure using a vacuum oven,optionally with heating to accelerate the drying process. 2.0 mL ofinternal standard solution (0.513 mg/mL propyl benzoate, Aldrich Cat#30,700-9 or equivalent, in 1,2-dichloroethane, Aldrich Cat #15,478-4 orequivalent) was then added to each dry sample, followed by 1.0 mL of 20%(vol/vol) HCl (EM Science Cat # HX0603P-1 or equivalent) in n-propanol(Aldrich Cat #29,328-8 or equivalent). Samples were then sealed withpolytetrafluoroethylene film (Teflon tape or equivalent), cappedtightly, and incubated at approximately 100° C. for 3 hours withvortexing approximately every 30 minutes. Samples were then cooled toroom temperature. An aqueous extraction was then performed by additionof 2 mL deionized water to each tube, vortexing for 10-20 seconds,allowing the phases to separate, and removal of the aqueous (top) phase.The aqueous extraction was repeated a second time, then the organic(lower) phase was transferred to a GC vial. Calibration standardscontaining between 0.2 and 10.0 mg/ml sodium 3-hydroxybutyrate (ICNBiomedical Cat #100964 or equivalent) were also prepared by the samemethod starting with the step of evaporation to dryness, i.e., thesodium hypochlorite digestion was omitted. Each sample and calibrationstandard was then analyzed using the Hewlett Packard Model 6890 GasChromatograph System.

The Hewlett Packard Model 6890 Gas Chromatograph System was operatedwith the following parameters: Sample Inlet: GC; Injection Source: GCALS; Mass Spectrometer: Disabled; OVEN: Initial temp.: 50 C (On);Maximum temp.: 325 C; Initial time: 2.00 min; Equilibration time: 0.50min; Ramp #1 Rate 7.00, Final temp. 120 C, Final time 0.00; Ramp #2 Rate18.00, Final temp., 280 C, Final time 2.00; Ramp #3 Rate 0.0 (Off); Posttemp: 0 C; Post time: 0.00 min; Run time: 22.89 min; BACK INLET: Mode:Split; Initial temp: 275 C (On); Pressure: 12.96 psi (On); Split ratio:10:1; Split flow: 11.0 mL/min; Total flow: 13.1 mL/min; Gas saver: On;Saver flow: 20.0 mL/min; Saver time: 2.00 min; Gas type: Helium; COLUMN2; Capillary Column; Model Number: HP 19091S-433; HP-5MS 5% PhenylMethyl Siloxane; Max temperature: 325 C; Nominal length: 30.0 m; Nominaldiameter: 250.00 um; Nominal film thickness: 0.25 um; Mode: constantflow; Initial flow: 1.1 mL/min; Nominal init pressure: 12.97 psi;Average velocity: 27 cm/sec; Inlet: Back Inlet; Outlet: Back Detector;Outlet pressure: ambient; BACK DETECTOR (FID); Temperature: 280 C (On);Hydrogen flow: 40.0 mL/min (On); Air flow: 450.0 mL/min (On); Mode:Constant makeup flow; Makeup flow: 15.0 mL/min (On); Makeup Gas Type:Helium; Flame: On; Electrometer: On; Lit offset: 2.0; SIGNAL 1; Datarate: 20 Hz; Type: back detector; Save Data: On; Start Save Time: 4.00min; Stop Save Time: 22.00 min; Zero: 0.0 (Off); Range: 0; Fast Peaks:Off; Attenuation: 0; POST RUN: Post Time: 0.00 min; Front Injector: Noparameters specified; BACK INJECTOR: Sample Washes: 0; Sample Pumps: 2;Injection Volume: 1.0 microliters; Syringe Size: 10.0 microliters;Nanoliter Adapter: Off; PostInj Solvent A Washes: 5; PostInj Solvent BWashes: 5; Viscosity Delay: 0 seconds; Plunger Speed: Fast; PreInjectionDwell: 0.00 minutes; PostInjection Dwell: 0.00 minutes.

A standard curve was fitted to the calibration standards by linearregression analysis using multilevel calibration with internal standard,resulting in the equation:

y = mx + b Where:$y = {\frac{{Area}\mspace{14mu} {PHB}}{{Area}\mspace{14mu} {Istd}} = {{Area}\mspace{14mu} {ratio}}}$$x = {\frac{{Amount}\mspace{14mu} {PHB}}{{{Amount}\mspace{14mu} {Istd}}\;} = {{Amount}\mspace{14mu} {ratio}}}$Istd.  is  the  internal  standard m = slope b = y-intercept

PHB content of the samples was then calculated using the followingequation:

${{Amount}\mspace{14mu} {PHB}} = {\frac{\left( {{Area}\mspace{14mu} {{PHB}/{Area}}\mspace{14mu} {Istd}} \right) - b}{m} \times {Amount}\mspace{14mu} {Istd}}$

Results

The presence or absence of PHB was confirmed using gas chromatography(GC). Diutan samples from strain S657/pS8 contained an average of 4.0%PHB by weight (Table 4). In contrast, PHB was undetectable in foursamples from each of two independent diutan preparations from strainPDD3/pS8 (Table 4). These results indicated that strain PDD3/pS8produced diutan containing less than approximately 0.05% PHB by weight(the estimated detection limit of the method).

As discussed above, abolition of PHB production by deletion of the phaCgene resulted in severe metabolic deficiency, poor growth, and greatlyimpaired diutan productivity. These results provide further confirmationof the unexpected finding that the diutan productivity and yield of aphaC deletion strain can be greatly enhanced by introduction of aplasmid containing genes involved in diutan synthesis, even though PHBproduction has not been detectably restored.

TABLE 4 Confirmation of absence of PHB by Gas Chromatography. SampleWeight Calculated PHB Strain (mg) (mg) Wt % PHB S657/pS8 39.8 1.68 4.2239.8 1.65 4.16 36.0 1.39 3.86 36.0 1.36 3.79 PDD3/pS8 33.1 n.d. n.d.(Preparation #1) 33.1 n.d. n.d. 38.0 n.d. n.d. 38.0 n.d. n.d. PDD3/pS838.5 n.d. n.d. (Preparation #2) 38.5 n.d. n.d. 38.0 n.d. n.d. 38.0 n.d.n.d. n.d.: Not detected. The limit of detection was 0.05% by weight.

Example 5 Diutan Filterability for Enhanced Oil Recovery ApplicationsMethods

Diutan fermentation broths were clarified with cellulase and recoveredas described in Example 3.

Filterability studies were performed on 0.04% diutan rehydrated inseawater. The diutan solution was passed through a 47 mm diameterNUCLEPORE™ filter (track-etched polycarbonate membranes havingstringently controlled pore size, available from Whatman, Inc.,Piscataway, N.J.) of the indicated pore size using a flow pressure of 20psi. The time for each 200 ml of the diutan solution (1 or 2 literstotal) to flow through the filter was measured with a graduated cylinderand a stop watch.

Results

In this example, the filterability of enzyme-clarified, rehydratedproducts from the PDD3/pS8 strain were compared to enzyme-clarified,rehydrated products from the 5657/pS8 strain. Enzyme-clarified diutanpreparations were filtered through NUCLEPORE™ filters of the indicatedsizes, and the volume filtered is shown as a function of time (FIGS.3A-3B and 4A-4C). Clogging of filters is indicated by lines tendingtowards vertical on the graphs, showing that little additional volumewas passing through the filter as time passed. Two preparationscontaining PHB made from strain S657/pS8 were poorly filterable,clogging filters of 5 microns (FIG. 3A) and 3 microns (FIG. 3B) beforeone liter could be filtered. In contrast, PHB-deficient diutanpreparations made from strain PDD3/pS8 showed improved filterability.Two out of three preparations were filterable at 3 microns (FIG. 4A andFIG. 4C), while the third was filterable at 5 microns (FIG. 4B).Together, these results indicated that the PHB-deficient strainsproduced diutan with improved filterability.

Example 6 Description of Plasmids pS8 and pX6

The plasmids pS8 and pX6 are as previously described in U.S. PublicationNo. 2008/0319186. In brief, these plasmids were obtained by screening anATCC 53159 genomic sequence library (in cosmid cloning vector pLAFR3)for clones able to restore polysaccharide production in the nonmucoidmutant (GPS2) of S. elodea ATCC 31461 or a nonmucoid mutant ofXanthomonas campestris. Plasmid inserts were end-sequenced and/orshotgun sequenced. A map showing the genes contained in complementingplasmids is shown in FIG. 5. The pS8 insert DNA sequence is provided asSEQ ID NO: 1 (FIG. 6), and the pX6 insert DNA sequence is provided asSEQ ID NO: 54 (FIG. 7). Predicted gene functions were designated basedon homology to other genes in public databases. Genes contained inplasmid pS8 and pX6 and their predicted functions are listed in Tables 5and 6, respectively. Pursuant to the Budapest Treaty for theInternational Recognition of the Deposit of Microorganisms, strains ofE. coli containing plasmids pS8 and pX6 have been deposited with thePatent Depository at the American Type Culture Collection at 10801University Boulevard, Manassas, Va. 20110, and are available as depositnumbers PTA-10102 (deposit date Jun. 2, 2009) and PTA-10103 (depositdate Jun. 2, 2009), respectively.

Plasmid pS8 contains the genes dpsS, dpsG, dpsR, dpsQ, dpsI, dpsK, dpsL,dpsJ, dpsF, dpsD, dpsC, dpsE, dpsM, dpsN, atrD, atrB, dpsB, rmlA, rmlC,rmlB, rmlD, and orf7. Plasmid pX6 contains the genes dpsJ, dpsF, dpsD,dpsC, dpsE, dpsM, dpsN, atrD, atrB, dpsB, rmlA, rmlC, rmlB, rmlD, orf7,orf6, and orf5. Based on their homology to known genes, many of thegenes contained in these plasmids are predicted to be involved in diutanproduction. The genes in the genomic region from which plasmids pS8 andpX6 were derived (FIG. 5) include genes that encode the transferases forthe four sugars of the diutan backbone and the four genes fordTDP-rhamnose synthesis. Genes for secretion of the polysaccharide,dpsD, dpsC, and dpsE, were identified based on homology to genes forbiosynthesis of other polysaccharides. Two genes, atrB and atrD, encodeproteins homologous to proteins involved in protein secretion. Twogenes, dpsG and dpsR, putatively encode a polymerase and a lyase,respectively. Two genes, dpsM, and dpsN, encode polysaccharideattachment proteins. The insert in plasmid pX6 contained 17 genesincluding gene dpsB encoding transferase I (which initiates the firststep in diutan synthesis), genes for secretion and four genes fordTDP-rhamnose synthesis, but lacks the genes for transferases II, IIIand IV and the putative genes for polymerase and lyase. Plasmid pS8contains 20 genes of the dps gene cluster, including genes for all fourbackbone sugar transferases, the four genes for dTDP-rhamnose synthesis,and genes for secretion of the polysaccharide, including the putativegenes for polymerase and lyase, but lacks the genes of unknown function,orf5, orf6, and orf7.

TABLE 5 Genes contained in plasmid pS8. Start and end coordinates arerelative to the pS8 insert sequence contained in SEQ ID NO: 1. SEQ ID NOGene Amino Start End Name Description DNA Acid    2* 1054 dpsS (partial)homologous to 2 3 gelS  2738 1113 dpsG putative polymerase 4 5  48952898 dpsR putative lyase 6 7  5093 6031 dpsQ putative rhamnosyl 8 9transferase IV  7082 6111 dpsI unknown 10 11  7121 8167 dpsKbeta-1,4-glucuronosyl 12 13 transferase II  8164 9030 dpsL glucosyltransferase III 14 15 10467 9079 dpsJ unknown 16 17 11076 12374 dpsFunknown 18 19 12389 13306 dpsD polysaccharide export 20 21 protein 1334114687 dpsC polysaccharide export 22 23 protein 14687 15394 dpsEpolysaccharide export 24 25 protein 15405 16286 dpsM polysaccharideattachment 26 27 16270 16968 dpsN polysaccharide attachment 28 29 1845417060 atrD secretion protein 30 31 20637 18451 atrB secretion protein 3233 21229 22641 dpsB glucosyl- 34 35 isoprenylphosphate transferase I22757 23635 rmlA glucose-1-phosphate 36 37 thymidylyltransferase 2363224198 rmlC dTDP-6-deoxy-D-glucose- 38 39 3-5-epimerase 24202 25263 rmlBdTDP-D-glucose-4,6- 40 41 dehydratase 25263 26129 rmlDdTDP-6-deoxy-L-mannose- 42 43 dehydrogenase 26277 26146 orf7 (partial)unknown function 44 45 *First in-frame codon; the start codon is notpresent.

TABLE 6 Genes contained in plasmid pX6. Start and end coordinates arerelative to the pX6 insert sequence contained in SEQ ID NO: 54. SEQ IDNO Gene Amino Start End Name Description DNA Acid 1 336 dpsL (partial)glucosyl transferase 46 47 III 1773 385 dpsJ unknown 16 17 2382 3680dpsF unknown 18 19 3695 4612 dpsD polysaccharide export protein 20 214647 5993 dpsC polysaccharide export protein 22 23 5993 6700 dpsEpolysaccharide export protein 24 25 6711 7592 dpsM polysaccharideattachment 26 27 7576 8274 dpsN polysaccharide attachment 28 29 97608366 atrD secretion protein 30 31 11943 9757 atrB secretion protein 3233 12535 13947 dpsB glucosyl-isoprenylphosphate 34 35 transferase I14063 14941 rmlA glucose-1-phosphate 36 37 thymidylyltransferase 1493815504 rmlC dTDP-6-deoxy-D-glucose-3- 38 39 5-epimerase 15508 16569 rmlBdTDP-D-glucose-4,6- 40 41 dehydratase 16569 17435 rmlDdTDP-6-deoxy-L-mannose- 42 43 dehydrogenase 18288 17452 orf7 unknownfunction 48 49 19433 18618 orf6 unknown function 50 51 19751 20683 orf5unknown function 52 53

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While the invention has been described by way of examples and preferredembodiments, it is understood that the words which have been used hereinare words of description, rather than words of limitation. Changes maybe made, within the purview of the appended claims, without departingfrom the scope and spirit of the invention in its broader aspects.Although the invention has been described herein with reference toparticular means, materials, and embodiments, it is understood that theinvention is not limited to the particulars disclosed. The inventionextends to all equivalent structures, means, and uses which are withinthe scope of the appended claims.

What is claimed is:
 1. A method of producing a sphingan essentially freefrom polyhydroxybutyrate (PHB), said method comprising, culturing amutant strain of the genus Sphingomonas under conditions that facilitateproduction of the sphingan, wherein the mutant strain comprises: atleast one genetic modification that substantially or entirely eliminatesa production of polyhydroxybutyrate (PHB) and at least one geneticmodification that results in increased production of a sphingan, whereinsaid genetic modification resulting in increased production of asphingan comprises a genetic modification that increases the expressionof at least one gene involved in sphingan synthesis, wherein said atleast one gene involved in sphingan synthesis is selected from the groupconsisting of the genes contained in the plasmid contained in strainATCC PTA-10102 and the plasmid contained in strain ATCC PTA-10103;whereby the mutant strain of the genus Sphingomonas produces anincreased production of the sphingan that is essentially free of PHBrelative to a congenic strain containing the at least one geneticmodification that substantially or entirely eliminates the production ofPHB and lacking the at least one genetic modification that results inincreased production of a sphingan.
 2. The method of claim 1, whereinthe sphingan is selected from the group consisting of diutan, S-7,gellan, S-88, welan, rhamsan, S-198, NW-11, and alcalan.
 3. The methodof claim 1, wherein the sphingan is diutan.
 4. The method of claim 1,wherein said at least one gene involved in sphingan synthesis isselected from the group consisting of Sphingomonas genes dpsS, dpsG,dpsR, dpsQ, dpsI, dpsK, dpsL, dpsJ, dpsF, dpsD, dpsC, dpsE, dpsM, dpsN,atrD, atrB, dpsB, rmlA, rmlC, rmlB, rmlD, orf7, orf6, and orfs.
 5. Themethod of claim 1, wherein the at least one genetic modification thatresults in increased production of a sphingan is selected from the groupconsisting of: (i) an operable linkage of at least one gene involved insphingan synthesis to an ectopic promoter; (ii) an increased number ofcopies per bacterial chromosome of at least one gene involved insphingan synthesis; and (iii) any combination thereof, wherein each ofsaid at least one gene involved in sphingan synthesis are contained in abacterial chromosome or extrachromosomal element.
 6. The method of claim1, wherein the at least one genetic modification that substantially orentirely eliminates the production of PHB is a mutation thatconstitutively or conditionally inactivates or deletes a gene selectedfrom the group consisting the phaA gene, the phaB gene, and the phaCgene or a combination thereof.
 7. The method of claim 1, wherein the atleast one genetic modification that substantially or entirely eliminatesthe production of PHB is an insertion or deletion that inactivates thephaC gene.
 8. The method of claim 3, wherein the mutant strain of thegenus Sphingomonas is able to produce diutan at a rate of at least about0.15 g/L/hr or a yield of diutan of at least about 12 g/L.
 9. The methodof claim 3, wherein the mutant strain of the genus Sphingomonas is ableto produce diutan at a rate of at least about 0.2 g/L/hr or a yield ofdiutan of at least about 15 g/L.
 10. The method of claim 3, wherein themutant strain of the genus Sphingomonas increases the rate of productionor yield of diutan by at least about 50% relative to a congenic straincontaining the at least one genetic modification that substantially orentirely eliminates the production of PHB and lacking the at least onegenetic modification that results in increased production of diutan. 11.The method of claim 1, further comprising isolating the sphingan fromthe culture, wherein the sphingan produced from the mutant strain of thegenus Sphingomonas is clarified to yield less than 0.5% residue in a 15%HCl solubility and residue test, or less than 0.1 wt % PHB when measuredusing gas chromatography.
 13. The method of claim 3, further comprisingisolating the diutan from the culture, and dehydrating the diutan,wherein when rehydrated as one liter of 0.04% diutan in seawater canpass through a polycarbonate membrane filter in less than five minutesat a flow pressure of approximately 20 psi; wherein the polycarbonatemembrane filter is approximately 47 mm in diameter and has a pore sizeof approximately 3 microns.
 14. The method of claim 3, furthercomprising isolating the diutan from the culture, and clarifying thediutan to exhibit a sea water 3 rpm viscosity of at least about 40 dialreading, a sea water 0.3 rpm viscosity of at least about 37,000 cp, or alow shear rate viscosity in the presence of polyethylene glycoldispersant of at least about 3,500 cp.
 15. A method of producing asphingan essentially free from polyhydroxybutyrate (PHB), said methodcomprising, culturing a mutant strain of the genus Sphingomonas underconditions that facilitate production of the sphingan, wherein themutant strain comprises: at least one genetic modification thatsubstantially or entirely eliminates a production of polyhydroxybutyrate(PHB) and at least one genetic modification that results in increasedproduction of a sphingan, wherein said genetic modification resulting inincreased production of a sphingan comprises a genetic modification thatincreases the expression of at least one gene involved in sphingansynthesis, wherein said at least one gene involved in sphingan synthesisis selected from the group consisting of the genes contained in theinsert in plasmids pS8 (SEQ ID NO: 1) and pX6 (SEQ ID NO: 54); wherebythe mutant strain of the genus Sphingomonas produces an increasedproduction of the sphingan that is essentially free of PHB relative to acongenic strain containing the at least one genetic modification thatsubstantially or entirely eliminates the production of PHB and lackingthe at least one genetic modification that results in increasedproduction of a sphingan.
 16. The method of claim 15, wherein thesphingan is selected from the group consisting of diutan, S-7, gellan,S-88, welan, rhamsan, S-198, NW-11, and alcalan.
 17. The method of claim15, wherein the sphingan is diutan.
 18. The method of claim 15, whereinsaid at least one gene involved in sphingan synthesis is selected fromthe group consisting of Sphingomonas genes dpsS, dpsG, dpsR, dpsQ, dpsI,dpsK, dpsL, dpsJ, dpsF, dpsD, dpsC, dpsE, dpsM, dpsN, atrD, atrB, dpsB,rmlA, rmlC, rmlB, rmlD, orf7, orf6, and orfs.
 19. The method of claim15, wherein the at least one genetic modification that results inincreased production of a sphingan is selected from the group consistingof: (i) an operable linkage of at least one gene involved in sphingansynthesis to an ectopic promoter; (ii) an increased number of copies perbacterial chromosome of at least one gene involved in sphingansynthesis; and (iii) any combination thereof, wherein each of said atleast one gene involved in sphingan synthesis are contained in abacterial chromosome or extrachromosomal element.
 20. The method ofclaim 15, wherein the at least one genetic modification thatsubstantially or entirely eliminates the production of PHB is a mutationthat constitutively or conditionally inactivates or deletes a geneselected from the group consisting the phaA gene, the phaB gene, and thephaC gene or a combination thereof.
 21. The method of claim 15, whereinthe at least one genetic modification that substantially or entirelyeliminates the production of PHB is an insertion or deletion thatinactivates the phaC gene.
 22. The method of claim 17, wherein themutant strain of the genus Sphingomonas is able to produce diutan at arate of at least about 0.15 g/L/hr or a yield of diutan of at leastabout 12 g/L.
 23. The method of claim 17, wherein the mutant strain ofthe genus Sphingomonas is able to produce diutan at a rate of at leastabout 0.2 g/L/hr or a yield of diutan of at least about 15 g/L.
 24. Themethod of claim 17, wherein the mutant strain of the genus Sphingomonasincreases the rate of production or yield of diutan by at least about50% relative to a congenic strain containing the at least one geneticmodification that substantially or entirely eliminates the production ofPHB and lacking the at least one genetic modification that results inincreased production of diutan.
 25. The method of claim 15, furthercomprising isolating the sphingan from the culture, wherein the sphinganproduced from the mutant strain of the genus Sphingomonas is clarifiedto yield less than 0.5% residue in a 15% HCl solubility and residuetest, or less than 0.1 wt % PHB when measured using gas chromatography.26. The method of claim 17, further comprising isolating the diutan fromthe culture, and dehydrating the diutan, wherein when rehydrated as oneliter of 0.04% diutan in seawater can pass through a polycarbonatemembrane filter in less than five minutes at a flow pressure ofapproximately 20 psi; wherein the polycarbonate membrane filter isapproximately 47 mm in diameter and has a pore size of approximately 3microns.
 27. The method of claim 17, further comprising isolating thediutan from the culture, and clarifying the diutan to exhibit a seawater 3 rpm viscosity of at least about 40 dial reading, a sea water 0.3rpm viscosity of at least about 37,000 cp, or a low shear rate viscosityin the presence of polyethylene glycol dispersant of at least about3,500 cp.